Most biological analyses of gene function have focused on protein-encoding genes, which constitute a mere 1.4% of the human genome. The rest of the genome is largely unexplored. The recent discovery of conserved stretches of non-coding RNAs, including microRNAs (miRNAs), has revealed a previously unrecognized layer of genomic regulation. miRNAs 20-22 nucleotides in length, function post-transcriptionally to titrate the activity of at least one-third of the protein-coding genes in the genome. In some cases, miRNAs function as on-off switches for key pathways. In other situations, they function as a rheostat to titrate the activity of pathways in normal biology and in response to external stresses and stimuli. Although more than 450 human or mouse miRNAs have been identified, most of the knowledge of this novel class of RNAs has come from worms and flies. Targeted deletion in mice has been reported for only three miRNAs to date, with two of them having critical functions in cardiac biology. These early loss-of-function studies have revealed novel targets for intervention in human disease, and it is highly likely that disruption of additional miRNAs will be equally revealing. To catalyze discovery in this emerging and highly significant area of biology, we propose to ablate -75 evolutionarily conserved heart and lung-enriched miRNA genes in the mouse with an advanced strategy to genetically modify embryonic stem cells. These mice will be made available to the scientific community through a web-based mechanism to accelerate advances in virtually every aspect of heart and lung research. This is an ambitious effort, but we believe we have the unique tools and expertise to accomplish it. In addition, the payoff is potentially significant. We expect major breakthroughs in the understanding of this relatively unexplored portion of the genome. New mouse models of human disease could shed light on human diseases in which potential protein-encoding disease genes have been difficult to map within a given genetic locus. These include a host of cardiac and pulmonary diseases of development and post-natal maintenance and adaptation. To accomplish this project, we propose three specific aims. Specific Aim 1. To generate embryonic stem cells and mice with disruption of ~75 heart and lung-enriched miRNAs. Specific Aim 2. To complete an initial characterization of the miRNA deletion lines, including validation of disruption and analysis of endogenous miRNA expression. Specific Aim 3. To disseminate information on and coordinate delivery of miRNA-disrupted mouse lines to the scientific community. (End of Abstract)